NZ721405A - Process for extracting lipids for use in production of biofuels - Google Patents
Process for extracting lipids for use in production of biofuelsInfo
- Publication number
- NZ721405A NZ721405A NZ721405A NZ72140514A NZ721405A NZ 721405 A NZ721405 A NZ 721405A NZ 721405 A NZ721405 A NZ 721405A NZ 72140514 A NZ72140514 A NZ 72140514A NZ 721405 A NZ721405 A NZ 721405A
- Authority
- NZ
- New Zealand
- Prior art keywords
- thc
- broth
- rnd
- oil
- oleaginous
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 150000002632 lipids Chemical class 0.000 title claims abstract description 15
- 230000008569 process Effects 0.000 title abstract description 19
- 239000002551 biofuel Substances 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 37
- 230000004151 fermentation Effects 0.000 claims abstract description 37
- 244000005700 microbiome Species 0.000 claims abstract description 27
- 238000010438 heat treatment Methods 0.000 claims description 13
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 210000004027 cell Anatomy 0.000 claims description 7
- 230000009089 cytolysis Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 5
- 238000009826 distribution Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 235000000346 sugar Nutrition 0.000 abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 31
- 108090000790 Enzymes Proteins 0.000 abstract description 16
- 102000004190 Enzymes Human genes 0.000 abstract description 16
- 210000002421 cell wall Anatomy 0.000 abstract description 6
- 239000000284 extract Substances 0.000 abstract description 6
- 102000001696 Mannosidases Human genes 0.000 abstract description 5
- 108010054377 Mannosidases Proteins 0.000 abstract description 5
- 238000005213 imbibition Methods 0.000 abstract description 4
- 238000005406 washing Methods 0.000 abstract description 4
- 239000004382 Amylase Substances 0.000 abstract description 3
- 102000013142 Amylases Human genes 0.000 abstract description 3
- 108010065511 Amylases Proteins 0.000 abstract description 3
- 235000019418 amylase Nutrition 0.000 abstract description 3
- 239000003921 oil Substances 0.000 description 61
- 235000019198 oils Nutrition 0.000 description 60
- 150000003839 salts Chemical class 0.000 description 22
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 18
- 239000002904 solvent Substances 0.000 description 18
- 239000000047 product Substances 0.000 description 17
- 239000002253 acid Substances 0.000 description 16
- 238000000605 extraction Methods 0.000 description 16
- 239000002028 Biomass Substances 0.000 description 10
- 239000000446 fuel Substances 0.000 description 10
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 239000007787 solid Substances 0.000 description 9
- 101100238304 Mus musculus Morc1 gene Proteins 0.000 description 8
- 239000010779 crude oil Substances 0.000 description 8
- 235000014113 dietary fatty acids Nutrition 0.000 description 8
- 229930195729 fatty acid Natural products 0.000 description 8
- 239000000194 fatty acid Substances 0.000 description 8
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 8
- 150000004665 fatty acids Chemical class 0.000 description 8
- 208000016444 Benign adult familial myoclonic epilepsy Diseases 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 208000016427 familial adult myoclonic epilepsy Diseases 0.000 description 7
- ZGNITFSDLCMLGI-UHFFFAOYSA-N flubendiamide Chemical compound CC1=CC(C(F)(C(F)(F)F)C(F)(F)F)=CC=C1NC(=O)C1=CC=CC(I)=C1C(=O)NC(C)(C)CS(C)(=O)=O ZGNITFSDLCMLGI-UHFFFAOYSA-N 0.000 description 7
- 150000002500 ions Chemical class 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000002203 pretreatment Methods 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- 241000195493 Cryptophyta Species 0.000 description 6
- 239000003925 fat Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 5
- 150000007513 acids Chemical class 0.000 description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 229910052751 metal Inorganic materials 0.000 description 5
- 239000002184 metal Substances 0.000 description 5
- 150000002739 metals Chemical class 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
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- 229910052708 sodium Inorganic materials 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 239000011324 bead Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 238000003306 harvesting Methods 0.000 description 4
- 229910052749 magnesium Inorganic materials 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- BKAYIFDRRZZKNF-VIFPVBQESA-N N-acetylcarnosine Chemical compound CC(=O)NCCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BKAYIFDRRZZKNF-VIFPVBQESA-N 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
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- 239000000178 monomer Substances 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
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- 238000010992 reflux Methods 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 description 2
- 241000609240 Ambelania acida Species 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101100398256 Mus musculus Kin gene Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 239000010905 bagasse Substances 0.000 description 2
- 239000003225 biodiesel Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- MHIGBKBJSQVXNH-IWVLMIASSA-N methacycline Chemical compound C=C([C@H]1[C@@H]2O)C3=CC=CC(O)=C3C(=O)C1=C(O)[C@@]1(O)[C@@H]2[C@H](N(C)C)C(O)=C(C(N)=O)C1=O MHIGBKBJSQVXNH-IWVLMIASSA-N 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- XURIQWBLYMJSLS-UHFFFAOYSA-N 1,4,7,10-tetrazacyclododecan-2-one Chemical compound O=C1CNCCNCCNCCN1 XURIQWBLYMJSLS-UHFFFAOYSA-N 0.000 description 1
- 241000531891 Alburnus alburnus Species 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229920001412 Chicle Polymers 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 240000002228 Cynara humilis Species 0.000 description 1
- 235000005921 Cynara humilis Nutrition 0.000 description 1
- 101100434929 Drosophila melanogaster csul gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101100001674 Emericella variicolor andI gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 240000001794 Manilkara zapota Species 0.000 description 1
- 235000011339 Manilkara zapota Nutrition 0.000 description 1
- 101100496087 Mus musculus Clec12a gene Proteins 0.000 description 1
- 229910003251 Na K Inorganic materials 0.000 description 1
- -1 NaCi Chemical class 0.000 description 1
- 101150034459 Parpbp gene Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 101000994634 Rattus norvegicus Potassium voltage-gated channel subfamily A member 1 Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 244000138286 Sorghum saccharatum Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- 102000005262 Sulfatase Human genes 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000004380 ashing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- VEVRNHHLCPGNDU-MUGJNUQGSA-O desmosine Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(CC[C@H](N)C(O)=O)=C(CCC[C@H](N)C(O)=O)C(CC[C@H](N)C(O)=O)=C1 VEVRNHHLCPGNDU-MUGJNUQGSA-O 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000003502 gasoline Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 244000062645 predators Species 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 108060007951 sulfatase Proteins 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
-
- C—CHEMISTRY; METALLURGY
- C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
- C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
- C10L1/00—Liquid carbonaceous fuels
- C10L1/02—Liquid carbonaceous fuels essentially based on components consisting of carbon, hydrogen, and oxygen only
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/04—Pretreatment of vegetable raw material
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/12—Production of fats or fatty oils from raw materials by melting out
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/063—Lysis of microorganisms of yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
Methods and systems used to extract lipids suitable in production of biofuels from a fermentation broth may include using heat to pre-treat the fermentation broth in order to more easily extract a product from oleaginous microorganisms in the broth. Additionally or alternatively, a combination of enzymes including amylase, 1-4 mannosidase, and 1-3 mannosidase may be used to break down cell walls of the oleaginous microorganisms. Residual broth water may be recycled and used as imbibition water for washing a process feedstock to extract sugar.
Description
Methods and systems used to t lipids suitable in production of biofuels from a fermentation broth may include using heat to pre-treat the fermentation broth in order to more easily extract a product from oleaginous microorganisms in the broth. Additionally or alternatively, a combination of enzymes including amylase, 1-4 mannosidase, and 1-3 mannosidase may be used to break down cell walls of the oleaginous rganisms. al broth water may be recycled and used as imbibition water for washing a process feedstock to extract sugar.
NZ 721405 PROCKSS FOR EXYRACYIING LIPlIDS FOR USK If% PROMJCYlON OF 'KJLS APPLICATIONS CROSS-REFERENCE YO RKY..AYKD This Rpplllcation cllalms bcncfit of U.S. provisional patent Rppiicatlon Serial No. 6i/9i8„850filed December 20„201I3.
NAMKS OF YHK PARYIKS YO A WHNY RKSKARCH AGRKKMKNY For purposes of prior art determination, a joint ch agreement was executed betweeli BP Blofucls UK Limited Bn«i MBTtck BEoscicQccs ation on Dcceinbci i 8, 2008 In thc field of blofucis. Also (ox pul'poses of pliol' Mt determination, 8 10IQt «icvclopQlcDi BgI'ccnlcnt was l0 executed between BP Biofueis UK Limited and Martek Biosciences ation on August 7, 2009 in the field of biofuels. Also for purposes of prior art dctemliiiation, a join( development lcQt was executed bctwccn BP Blofuels UK Limited Bnd DSM Blobascd PTO«lucis Bnd Scxvlccs B.V. 0EI nbcI' l, 20i2 IQ thc field of biofucls.
CAX, FrK3.D Thc InvcntloQ relates to Encthods Bnd s directed to cxtlRctlng rnatcx'lais foI' biofuc1ls production. Aspects of the lnvcntlon rclatc to extracting TEIRtcxials fxonl Olcaglxlous ml cI'0organEsExis.
BACKGROIUEEE tt A number of technologies fol' converting fcc«1lstocks Into biofucls have bccxl dcvciopc«i.
Howcvcx„cvcQ with thcsc R«ivanccs in technology, thcl'c xclnMDs 8 nccd MMl 8 desirc to IrnpTovc ccoEloExlic vlabllEty for coDvcrsion of renewable cai'boQ s to fuels.
Vegetable-Oil-derived biofucl DEay have bcTEcfits, such Rs being renewable. biodegradable, nontoxic, Bnd, IQ ccxtMD cases, containing neither suifUIT nox Brolnatics. Bur onc potential «1llsadvMltagc of ble-oil-derived blofuci is high cost, most of which ls duc to thc cost of the vegetable oil feedstock. Therefore, the economic aspect of biofuei production has bccQ Bi least sorncwhat limited by lhc cost of thc vcgctablc oill raw Inatcxlais Bs well Bs thc inrntcd supply of thc vcgciab1lc oil I'Bw Inatcrials.
Llpids fox usc ln QUltritional cis can bc pr'0«iuccd In organlsxrl, Manufactuling 8 hpld m algae for example xnay mclude growmg the algae drying lt and extractmg the IQixacciiular llpids froTD lt. Extracting material from within thc microorganism can bc difficult.
SETAElarly, vcasis, inclluding oleaginous yeast, , have polysaccharide cell walls to protect thcin from cnviromnciital stl'csscs„such Rs shear forces, Oslnotic ELDbRlanccs, dcsiccRtion, ors, and the like, The protective ceH wall can xnake it ult to harvest intraceHular TnctRbolitcs„such 8S lipKls, ELI Oleaginous yeast thR't cM1 bc converted Ento biofuei.
Convertiiig sugar to biofucl using heterotrophic Organisnis is possible wiith RA aqueous Or soivcQt cxtiaction section, EB which pM ts of thc ol'gaBlsnis Rrc dissolved EQ water ol' another solvent, thus cARbling thc product llpids to bc rcniovcd MKl vcrcd (ill cctiv froni 8 fcrmcntarion broth. Product Enay bc iccovcl'cd fionl lntcI'Dal cornp8rilncQts Of thc Oleaginous organlsxn by conibinations of mechanical, Eal, osinotic, and cnzyniatic foiiccs., resulting lB 8 l0 Biultl-phase product stl'cRID ting of light lipids, 61lclipidatcd bioniass, RQd RqucoUs rcsiduc Rnd other cell IcsiclUc. OQcc-through ploccssing often results in 8 considerable v"Rstc Rnd/01 co- product streani(s).
Thcrc ls 8 Acc(l Rnd 8 dcsilc for Encthods RQd ns foI' cxtl'8ctlng Biatcrials From OlcaglQous IDEcroorganisnis that I csul't ln 8 high yield Of Inatcrial cxtl'8ctcd. Using Bon- l5 solvent/aqueous cxitraction tcchnlqucs. Thcrc is 8 further Aced Mld desirc foI' LDcihods Rnd systcxQs for cxtractlng IBRtci'lais floDI Oleaginous LAEcroorganlsxns that rccyclc al pi'occss s„rather than relying on once-through processing.
SOU MMARV Thc Hivcntilon lclatcs to ds RLEd ls fol extracting Bmtclials fI'01TI OlcaglnoUs DilcroorganlsLDs, . Rs wclll Rs Inethods Rnd systcnis for cing bioFucls froLD thc extracted According to ccrtalQ cniboc1IEDEents, tcELIpcraturc may bc used 8s 8 prc-trcatxAciit step to iEDprovc thc cxtI'action yield of i froxn 8xl olcagiQ0Us organisBl. Mol'c pMticul8x'lv, 8 Lnethod of extracting lipids suitable in production of biofuels fmxn a whole fernientaiion broth Lnay Include icatiBg ihc whole Fcrincniation bioth by g thc brolh to 8 temperature greater than about 90'C, such as between about 90'C and about i 50'C, or between about l 00'C bout l50"C or between about l l0'C Rnd about l 50'C Or betwccxi about l20'C aiiidabo l30 C, ol' bctwccQ Rbout l30 C Rnd about 130 C, whcrcui thc bToth con'tains oicRglnous InicrooI'gREiislns, Rnd subsequently cxtx'RctlBg 8 product froxn the oic8glBous IBECT001'gRBisnls.
Thc whole fcrlncntation bro'th Inav bc hc8tcd for Bmrc Xhan Rbo'Ut' 3 hoUTs. hi ccrtaiQ cniboc1IEDIents, tEGlc spent by thc whole FcxlncBtatloD bToth coniIsiinlng thc 01cagiB0US microorganisms between 45'C and 80'C may be lnimmized by heat~ng the whole fernicntation bfoth coQtaimDg thc oleaginous ofgRDlsrns ffoin 45'C to 80 'C ED less thBQ 60 minutes.
AdditionaHy or altefnativeiy, the whole fermentation broth rIMy be heated at an average rate bctwccn about 0.l Bnd about 80 dcgfccs CclsiUs pcT minute, in this ss, pH of thc whole fermentation bfoth. DMy bc Rd)usted by adding cfthcr Rn acid 0T 8 base, in further embodiEIIents, the whole fermentation broth may be cooled to greater than about 60"C, of gfcatci than about !O'C, 0I gfcatcl &an about, 80'C, 0T gfcatcf tllian Rbout 85'C, or greater than about 90'C io RHow further isotll~ermal (constant temperature) procesSin, such as by Bpplvlng cRl disDlption. Thc whole fcffncntatloD broth QMy bc cooled Bt Bn Rvclagc i0 rate between about i and about 80 degrees Celsius per minute. The whole fermentation broth IQBy bc agitated Bt an lfnpcHcl' tip spccd n Rbout. i0 CIQ pcf secoiicll Rnd about 240 cnl pcI' second. F0HOwlng the heating, tile whole fcrmcn:tatlon bl 0th Enay bc dried. ln certain embodiments, duFillg the pfc-treating, 8 prcssure bctwccn about i0 psl and.
Rbout i 50 psi, 0T bctwccQ about 20 psl RQcI. about i 50 psl, 0F bctwccD BboUi 30 psl Rnd about l50 i5 psl., of bc(wccn aboUt 50 psi Bnd about i50 psi niay bc Enainiained m 8 systcDI coiiiaining 'thc DUFEng thc pfc-tf catmg, salts mRv bc pI'cscnt 1Q 8 svstclfi contRlning thc whole tation broth, irlg in an ioflie strength estimatecl behveen about 0.0i M andI about 2.0 M in 'thc svstcm. The whole fermentation broth Enay lnchKlc 8 crude sugar source and/of 8 xvatcf soul'cc associated with salts Bnd lons Bt 8 coQccntf'ation gfcRtci tbRD 0.05 g/L. Yhc salts Bnd lons may iinclude Na, K, CB.. Mg. ZD„MO, Cu„Mn„chlorides, sulfates, phosphates, nitrates, and coIQblDKtlons thcfcof, These salts BDd lons Dlay build up to 8 conccntl'Rtlon of 0.5 to 40 g/L.
AdditioDRHy, thc salts Bnd lons that 81'c already pl'cscnt. GMy help with I'ccovcfv of Bfl oil phase bv promoting ccQcc, i|occulatlon, density clMngc, Bnd/of dcstablilzing ihc cfnulsion foITDcd when thc product ls fcllcascd ffoln the oleaginous rganisms iD RDECBl Bnd/ol Methods herein DMy fGTthcf Include sub)ecting the oleaginous DzicT001'gBQlsms to lysis, resultmg in an oil body and ceHular debris le size distribution in which at least 80'/o, or at least 9&",of a volulne of eleased product cil bodies and ceHula debr . ha e a size greate than 0, i UIQ ln (cr. Additionally, thc OH Bnd ccH debris droplets ol bodies Fnay bc fccovcTcd as 8 continuous phase by mixing Bt RD ilnpcHcI' tip speed of gI"eatel' than i20 c]Ti/s.
After bI'cRklng dowB t4c nicaglQ0Us ccH wallis, IDtfaccHUIRT Qlctabniltcs EBCIUdlng ilplds„ for example, may be harvested from ihe oleaginous ceH walls. The ceHuiar htes may bc coQvcFtcd. into binfuci, such 8S bio-«icrived «bcsci. AB 8«iucous tion cNucnt fcn!RHling after haisesting the eHuiar Inetaboiites may be recycied. The recycied extraction water Hlay bc Used Rs EIDblbltlon wRtcx' foI' wRshlng 8 pfnccss fcc«istock to cxtTRct sugar.
I'OHowlBg t4c prc-tl'caBDg, thc fcxxncBtailon bro(h BIRy bc ssUI'1/c«1 Rnd conic«1 io cnnccQtl'atc solids ln t4c broth prior to fUxt4cF sslBg. onaHy nf alternatively„ lg thc pr'c-tlcatlng, cvapof8tofs or drycfs coul«1 bc included to generate 8 conccntfa(cd wet broth or a dry Enixture with ceHs. A solvent may be added to ihe dfy ceHS or in the iysed concentrated fermentation broth to form a mixture, The solvent may include hexane„dodecane, dccanc, diesel, onc ol' lilofc alcohols, 01' combinations thereof. Thc inlxtUI'c of thc ivsc«1 feITnentatinn broth and ihe solvent may be agitated to contact and extract oil floin ihe oleaginous InlclooI'gRnisIDs Thc solvent 8Qd thc 011 BMy bc scp8TRlc«1 froID thc Ilvscd fcFIncntatioQ blot4„ such as by using a centrifuge. The solvent and the OH may be reacXed io convert at least a poition of t4e nil into a fuel component, Furthermore„ the solvent and a remainder of the Oil xnay be converted iDtn 8 fuel cornprEslng 8 biohlci. Thc spent bfntll Inav bc usc«1 as fcrtlhzcr fnl cx'ops, animal fee«L yeast extract, yeas( hydrolysate, or a source of carbon/nutrlexlts. in ccf(aln cmbodlmeDXs, Xhc whole fcTrncntailnD brot4 contamHig thc oleaginous microorganisms may include a sugar cck. The whole ferlnentatinn bmth containing the oleaginous rnicmofganisms and the sugar feedstnck may include about 50 tn about 250 grams nf hpld pcT iltcl' of fern!enter broth, about 0 to about 50 grains of s'UgRT pcl' iltcl of fcfmcntcl' broth„ about 0 to about 40 grams nf salt per liter of f'ermenter broth„and about 10 to about 100 grams of Hpid-free dry biomass per iiiex of fefmenter broth.
AccoTdlng to sonic cinbndlmcn(s, as part of the prc-trcatinent, t4c method DMy fufXhcf include pastcUI'lzlBg 8 whole fcllncntation bro'th coQtRH1Hlg thc OlcagnloUS DlicFoorgaQlsDls, suc4 as by hcatlDg Ilhc vvllloic tation 410th to about 40 C to about 80 'C fnr about 1 xnlnutc Up to 840Ut 3 40UTS. 1Q contrast, «1luring thc 'cRtxQcnt hcatEng, thc whole fcITncBtatlon bfnt4 Hlay bc held Rt. 8 temperature bctwccn about 90'C RQd RboUt 150 C., nr bctwccQ about 100 C Rnd about 150'C„orbetween about 110"C and about 150"C, or between aboUt 120'C and about 150'C, or between about 130"C and about 150'C f'Or about 30 s to about 18 hours, or morc thaD 3 40urs to about 18 hours„ol. Inofc thRQ 3 40UIs to Rbout 8 40UIs. 'ihc whole fcrnlcntatlon broth IDav bc stlTTcd dui'Eng thc heating interval. AD RCK1, 8 base„or both Bn acid BYEd 8 base may be added to the wholle fermentation broth.
The whole fermentation broth Inay be passed through a bead miH„orifice plate, high shear mixer, or other shear or mechanical disruption device once„ twice, or more, ln certain enlbodjinents thc whole fcrnlcnYatjon brorh may bc stjrrcd jn 8 vessel Rt BboUY 70 l 00 C optloDRHv includEng reflux for Bbo'ut 1 to BboUt 60 ho'Urs, AddltlonBHy, 8 salt such Bs NaCl, KCl, KZSO4, or NBZSO4 may be added Yo the whole fermentation broth in the vessel oi alternatively may be produced iLI situ, for exalnple, by adding NaOH or KOH, plus HLSO4. Up to about 2 yo by wcEght of thc sallt, Ynay bc added fol" cxalnplc AD acid OY' 8 base QMv bc added Xo Bc1ljust 8 pH of thc wllolc fcYYQcntatioQ broXh ln thc vcssci to bctwccEE about 3 Bnd about 11. 'Ifhc heat gcnclBtc«1 froYD Yhe natloQ Of Rci«ls Rml bases 1 above coul«1 also colltrlbutc to Tc«iuclng thc cQcigy lcqull'c«1 fol hcRtEng thc bloth. Thc llpl«ls QMy bc scpaiatc«l froin thc R«iueous fcITDcnta'tlon bloth through RQ Rppropl'late solid-liquid-liquid separation schcmc that may include one Or more steps such as gravity separation, hydrocyciones„ filters, and/or cclltrlfugcs. Oil that is less than 20~/o free fatty BCKls xnay bc tc«l from thc whole fermentatKIQ broth Yhrough fugation. This method of extracting hplds suitable En the production of microbial oil Ynay result in an oil that is RTYjfjcjaHy lower in metals, as the aqueous extraction pToccss txBtcs thc metals nl fcrmcLEtatioLE broth Bs coxQparc«l to thc 011 by 8 ratio of Bt lcasi 2, Thc Yncthod lnay ful'thcl' lilcilKlc I'ccvcllng thc s solids with thc TcsKluai l w ater, The oleaginous microorganisms Lnay include Rt least 40'r'a by weight fat. For exainple, the oleaginous Inicroorganisms may be Oleaginous yeast cells.
According Eo certain embodiments, 8 coixlblna. tlon of cnzynics including Rxnylasc, 1-4 Lnannosidasc, Rnd 1-3 sidasc Inay bc used Xo brcak «lown oleaginous ccH wBHs of thc noUs microorgREEislQs. Thc coEIibLnatlon Of cnzvIDcs QMy further e Rt least onc xy cQzyIDc, QREYlclv sulfatasc, . protcasc, chitlnRsc, ol' Rnv coxQbilMtl. ons of these enzymes.
The amylase may be ic for alpha 1-4 linked. glucose, The coinbinatjon of enzymes may C betWCCQ RbOut 5 ./6 RQd about 30/g by Vcei&ht REQyiaSC betWCCQ about 5 /o arid about 45,/o bv weight 1-4 mRMlosidasc, bctwccn about 0 c/o RIKl BboUt 45 r/o by weight 1-3 mannosidasc, oi BDy coQlbinatloD of these palaxnctcl's fhe cnzyinc coIlibinRtlon Qlay also include clt least onc auxiHary enzyme, such as sulfatase, protease, chitinase, or any combination of these enzymes.
The enzyme combination may be used wiith Sj3oridio/3o/us parriroseEES MK29404. As oned, RAcY' brcBklng down thc 01caginous ccH waHs, HltraccHUiar mctaboiitcs ii1lcluding llpKls„ fol example, may be harvested from the oleaginous ceH wa1lls.
According to certain embodiments& 8 Method of cxtY'acting liplds suitable lYE production of biofucis froEYl 8 whole fcITncntatloQ broth may include Rpplvlng 8 combination of cnzymcs to thc whole fermentation broth containing oleaginous microorganisms to break down ceH waHs of the Oleaginous organlsms, whcrclQ the cnzyTncs include aniylasc, 1-4 mannosidasc& Rnd 1-3 Mannosldasc& Rnd subscqucntiv extracting 8 produc( fl'oin tlhc olcaglQous InlcroorganisIns. Vhc combination of enzymes may further include at least one auxiliary enzytne such as ase, pY'oilcasc, asc& or BQv comblna(10YE of these enzymes. Thc BniyIIRsc MRv Ilc specific for alpha 1-4 Hnked glucose. The colnbination of enzymes may iinciude between about 5/o and about 30"/o by weight RQ!ylasc& n about. 5 ro Bnd Rbout 45 N& II3y weight 1-4 sldasc& bc(ween BboUt 5 /g Bnd about 45 /o by weight 1-3 Inannosidasc, 01 anv conlblnatEOII oj these parameters.
The method. Ynay further include harvesting mtraceHular Tnetabolites, such as hpids, from ihc 01caglnous Microorganisms after I31'cakmg dowQ thc ccH waHs. Thc EEY(raccHular IQciabolltcs Mav bc convcrtcd into biofuci& such Rs bio-derived diesel. AdditlonaHy& RQ aqueous cxtractloEY cfflUcnt Tclnaining after harvcstirlg thc intraccHuiar Enetaboiltcs EYERy I3c lccyclcd. 1he cd extraction water may be used as imbibition water for washing a process feeclstock to extract SUgBT. ing to certain cTnbodiMcnts, 8 Incthod of cxtractlDg ilpKis sultall31c 1D production of biofucis from Bn aqueous fcIYYicntBtloQ bl'0th Inay inchKlc cxtrRc'ting s froln 'the RcJucous fclrncntation bl"oth& whcTcin thc broth contains nous mlcroorgRnlsEYls or sugarcane& or both oleaginous microorganisms and sugarcane, leaving s s and residual bioth water, and Using thc Tcslduai broth water Rs 1EYEII31I31tEOD water fol' wRshiElg 8 pi'occss fccdstock to extract sugar. Thc IQcthod lnay further Enciudc pastcuI'izing ihc s ntRtioQ broth, such. Rs by heating thc Rquco'Us fcrnlcnYatlon broth to Rbout 40 C to about 80 C for Bbout 1 minute Up 'to BII30ut 3 houTs. in ccrtalQ cmbodimcilts& thc method Inav include g thc aqueous fcimcntation broth to 8 icnlpcraturc between Bbout 90"C Rnd about 150 C, 01 bc(%veen about. 100"C and about 150"C, or between about 110'CBnd about 150'C, or n about 120'C and about 150'"C,cr between about 130"C and about 150'C and holding the broth withi~ the selected I'ange for' BboU't 30 minutes to 8130Ut. 18 hoUrs, OI' Inorc than 3 hoUI's to about 18 hours& ol nloTc than 3 houI's to about 8 houls. The aqueous fcrmcrltatioIE bl'0th IQRy bc stEFrcd dunQg lhc hcatillg lntclval. An acid, 8 bBsc, GT both Rn Rcl«l Bnd 8 bRsc Fnav bc added to the us fermentation broth. Yhe aqueous fermentation broth Enay be passed through a bead ImH or other mechanical disruption dcvlcc once, twlcc, or lrlol'c.
IIIRIKP DKSCRIPYION OF YHK DRJE %BEINGS The ac( OIQpanvlng dl'RwEQgs wl. lich Brc Incorporated ln 4End conhtl tutc 8 pRrt 0( thl 8 spcclfIcation, EHustrate embodiments 01 the EEEVCQEEGQ and, together with the descrlptlon serve to cxplMQ thc fcMUE."cs, Rdvantagcs, Bnd plcs of thc loQ, In thc «ll'BwEDgs; FLG. 1 ls 8 ploccss flow diagranE lllusiITRtlng onc mMnt of. Rn Bqucous cxtTRctlon s using temperature pre-treatment and including the production Of 8 yeast extract.
FKx 2 ES 8 s flow n1 EHUstrktlng onc cmbodlrncnt of dn Entcgrdited sugar-to- (hCSCl pl'ocCSS 1QCluding I'CCVC1C.
Es 8 pToccss tlow dl Bgil BTD EHuslratlng ME aqueous cxtractlon pl'occss Used m Example 2.
F76. 4 is a graphical representation of particle size distribution of released oil and ceH debris foHowiing lysis in Example 3.
F76. 5 Es 8 graphical Fcpl'cscntatlon 0$ partilclc slzc dlstl'Ebutilon of oil Rnid ccH debris foHOWlng 011 pl'OdUCt FCCovery 1IQ EXRTQplC 3, DKYAII, KD DKSCRIPYION Thc Envcntlon provwics ods Rnd systclns for cxtFRctlQg Inatcrlals fl'GIQ olcBglnous DllcroorgamsETEs, Rs wcH Bs methods RQ«l systems for pr'oducmg ls from the cxti'Rctcd materials. ProducEEGD of blofucls from micr'oorgamsIns Inay have EI1Miy ages over pro(iuctlon of blofuels from plants (Encludlllg OElsccds)„such as short hfc cycle, less 1Rbor Fcqml'crncnt, . ED«lcpcndcncc of season RQd clllnatc, BQd caslcl scale-up.
As des(Tibed in greater detail below, pre-treatment of fermentation broth before oil cxtractEOD by directly heating thc broth io Fclailvclv hEgh tcQ1pcraturcs can Increase the amount of oil cxtrRctcd from olcaglQous mlcrool'gRQlsirls via thcIIQ81 dcgradan()n of thc ccH wRH stluctUrc such tlhat pciilncabllltv ls EDcrcRscd BEEd oil CBQ dlffusc morc readily. ODBHy or Rltcrnatlvclv, 8 nRtlon of enzymes including Mnvlasc, 1-4 Enannosidasc, Bn(1 1-3 mannosidase may be used to brcak down oleaginous ccH waHs of the oleaginous xmcroorganisms, ln any of the methods described herein, the aqueous extraction CNuent rcmaEDEng Biter ilpld FcrnovRl may bc Eccyclcd to thc front-cnd sugar recovery opcI'Rilons.
As used herein, the terms "'pre-treat" and "pre-treatlnent'" refer to process steps thai are caned out on 8 JQlcrooJ'ganlsrn prioT to physlcBily scparatET!g Mly xnaicriais from within ihc Qllcroorgalllsm.
As used herei~„ ihe teJID "'renewable material'" preEerabiy refers to a substance and/or an ltcrn 'tl'lai hRs bccQ at least pM"tiaiiy dcJ'lvcd from 8 source Rnd/or 8 process capable oi bclng replaced Bi least ln pari by natural ccologlcal cycles BJld/or FcsouI'ccs. Rcncvu'able lais may y Include, for example, als, chemical intermediates, ts, adhesives, lubricants, monomers, Oiigomers, polymers, biofuels„biofuei interlnediates„biogasohlle„biiogasoline blcndstocks. blodlcscl. gx'ccn diesel, TcJEcvvall3ic diesel„blodlcsci blend stocks, blodlstEHatcs„ blochaT, blocokc, blologlcal oils, rene'svabic bulldlng materials, Bnd/0E thc llkc. As 8 ED0I'c specific e, the rcncv'able material ITlay lnchldc, vvithout II3clng ilDllicd io„anyOQc 0I' nlolc of thc vlng; methane, ethanol, Q-buiRnoi„ lsobutan01„2-buianol, fattv alcohols, lsobutcnc, Esoprcnolds, trlglyccrldcs„ ilplds„ fRity acids„ lactic acid,. acetic acid, propancdloi„butancdloi.
AccoTding to ccrtaln cmbodEIncnts, thc renewable 181 may EEEchidc onc 01' EDOI'c i components. For example, . the Fenev;able material may include an alcohol, such. Bs ethanol, buEBnol. or Esobutanol, 01' ilplds, ln ccl'JBIQ cmbodllncnts„ thc rcncwvabic Qlatcrlai can bc dcrEvcd trom 8 living organism„such Bs algae, bactcrEB, fuQgl, Rnd/or thc ilkc. AccoI'ding io ccriMQ mcllts, thc renewable Inatcrlai ls R llpld„such Rs faXty acids vvlth 8 Q cllMQ length Thc term "blofucll"' prcfcI'abllv Icfcls io oncnts Bnd/or stx'cREDs sultRI3ic fol Else Bs 8 fuel RQd/or 8 coEDI3ustioQ source dcI'lvccl Bt least ln part from renewable sources. The II3iofuei can bc susialnabiv ccll REEd/or have reduced Rnd/or Qo Qct caI'bon ons (total carbon cic) to thc RtEDosphcrc, such as vhcEE compared to fossil fuels. Accordixlg Eo ccrtaln IDcnts, rcncxvabic soul'ccs can cxciudc nmtcllals Flllncd or driHcd, such Bs froED ihc underground. In ccriMD cnlbodimcnts, renewable souxccs cMI Enciudc slngic ccH 0I'gRnlsnls, IQU1'tl-cell OI'gRnlsIDs, piaEEts, fungE, bactcrER,. algae, cultivated crops, Qon-cultivated crops. timber, Rnd/OF the ilkc.
According io ccFtaln clnbodiIYlcnis, thc rcncvvabic soux'ccs include Qlicroorganisnls.
Biofueis can be le for use as transportation fuels, such as for use in iandI vehicles, marine vehicles, aviation vehicles, and/or the like, More particularly, the biofuels /nay include gasoline„ diesel, jet fuel, ne, and/or the hke. ls can be suitable for use in power tion, such as g steain cxchaQgnlg CQergy with 8 sultabje heat tl ansfer Fnedia Dcncrallng syngas generating I'lydl'ogcQ Qlaklno icity, Rnd/or thc 1Ekc. According to ccTtanl cinbodi/Dents, thc biofucl is 8 13lcnd of blodicscl and pctrolcUIQ diesel.
Thc tcnrls biodicscl and bio-dcrivcd dlcscl, Rs used herein, Rlc used interchangeably and refer to components or strealns suitable for dlirect use and/or blencling into a diesel pool and, 'or 8 cetane supply dlerived fiorn renewable sources. Suitable biodliesel Ynolecules can include fattv RCK1I cstcrs. 810dlcsel can be Used En con/pression Egnlt/on engines„such as 1I0 automotive diesel internal conlbustion engines„ truck heavy duty diesel s„and, 'or the hke, ln the alternative, the biodiesel can also be used in gas turbines, heaters„boHers„and/or the hke.
According to certain elnbodilnents. the biodiesel andi/or biodlesell blends Ineet or comply with industrially accepted fue1 standards, such as 85„87„8l0,8l5, 820, 840, 860, 880., 899,9, 8I00. and/or the like.
The term "lipid, " as used herein, refers to oils, fats, waxes„greases„cholesterol„ glyccl/dcs, steroids, phosphatldcs, ccrcbrosidcs, f88' acids, fatty acid Fcfatcd co/Dpounds, Ylcrlvcd colnpoUBds, OYhcT 0Elv subst8Tlccs, Rnd/or thc like, Lipids typically Enclmlc 8. relatively high energy content, such as on 8 weight basis.
T1IM tcrIB "EBEcroorganisnz, as Used hcY'clB, refers to 8 lr/lcroscoplc 01'ganlsln, which FBRv bc 8 slng1lc ccH (Unicellular), 8 cc]! clUstcT, or 8 nlultlcclllular Fclatlvcly conlplcx organlsnl.
Microor~aQisrns can include algae fungi (i/lclluding vcast)„bacteria 3actcria protozoa and/or the like, In onc clnbodEIBcnt, thc nllclooIgRQlsIQ cRQ bc 8 single cell IncIBbcr of thc fungal IQ, such as 8 yeast, for cxarnp1c. Examples of OlcaglBous fuDgl that. can bc used Include, bUt RT'e Bot ILIBLtcd to„A'/YodofoFL//a /tigon/05// 01 Spof/dto/301//5 pal"/Yl"05et/5„as % cH Rs YncLBII3crs OI thC foiloWEng gCQCTR. LSPeP"g~lllAS C/tttdtd// (.FVPFOCOCC//5 LYC/3//t'OmfCe5 krtdO/YYPCOP5/5 Ft/5/tr/Lttt/, 680/P/CAltrt/, PYCAka, L/POPE/'Ce5, Mt/CO/', 5 eti/C/ll//t//I„PIC&M, PMttdOZJ/tlt/, OLL/5, R/LOdOYOP"t//C/, POF/d//tt/I, SPOFO&0/OM')&Ce5, SAP /iteMlkk, JOT/tlt/SPO/'/t, Tt'/cjlospo/"on, P /clceFA//N/ott/pce5, Far/"owtQ, ct/5, Rnd. Zpgol/pOYYY@c85. Morc par(icujarlv, the oleaginous fungi nlay LIYcllude, for cxaIQplc, RQQ of thc following" . .4/3/orrtckttrt/ cttrvtttttrt/, Cand/da QJ//COkt, Cartdtdrt h/CO/t/, Ca/Id/dr/ 0/COQUE/I/la, Ct///dtdt/ 5P., Cattdtda /t OP/Ca//5, WO 95462 PC T/US2014/071055 Capp/OCOCCMS Q&tdWS, Capp/OCOCCWS CBFVQEPLS, Capp/OCOCCBS /ef'f/CO jWS', Dej)QFOff/'))CeS enf/, PB')PCOp$/, '& VefP/Q AS, GeO/I/CAW PP/ CQfQ j)lLCJQfBm, GeO/fl CABPf/ CWCW/E)/dQf/ Eff/, Ge0/f/CAW ff/ AES/CftdQP'Bluff, GeO/f/CALEPP1 5/ jV/CO jQ, Ge0/fECABPf/ VBjgQf'e, BgpAOp/C j/EQ j)E/f/OPE//, LlpOPHpCE. '5 jtpOJef, I/pOplg~CCS Ofeft/Q AS, I./pOppt&&&'Ce$5&EQf /A+/, LlpOplpceS /e/fQSpO! OBS„P/CALQ EBeXtCEE/EQ„ NlOdOSpOftdtttm SpIEQefOCQP'pBI, RAOJOSpOftd/BPPE fOf'W jO/deS& 8AOdOEOft/ ji/ Sp ., EOfltk/ QttfQPL/EQCE/, RAOdO/OfW jQ dQ/ e/tef/5/5, RAOdOEOP" BjQ dtffjWef/5, RAOdO/Of/di/ gjB//nWS, Of'W jQ gjW/Vl/S„RAOJO/OfB jQ gP QC/ j/5, AAOJOEOflt jQ gfQfPll/11$, AAOdOEOfW jQ fftlfltt/I/, RAOJOEOfllkt PPEWC/ jQglMPSE/, Of W kl Vlt/C/ jQgEPEOSQ, WlOdOLOf BjQ f'Wj)fQ, Of BjQ /ef pePEOtdQ j/5, 8j/Od0/Op'W jQ /Oft/ jOEJCS, SpOfOj)0 jOmg&Ce5 Qjj)0f'WjpeSCef/5, S/Qffp/eM jjQ j)Opt j)LCO jQ, TOfBkESpOpQ l0 ikjj)fBejill, TOTBkESpOfQpfC/Oftef/SES&, TOfBjOpSES j/pOjefQ, TOfWp0515 Sp. , Tf'/CAOSpOP'Ofl j)eAMnd„ Tf/CAOSpOP OP/ j)l QSS/CQe, TP"/C j/OSpOfOP/ CQp//Q/Bm, TP ECAOS+OfOP/ CEE/Qf/eWPPE, Tf/CAOSpOf'OP/ ij0fftcs/tcwfft, 5&pOf'ofl jQt j)Qcjtll, Tf/cAOSpofofl joBj)tef'l, spOfof/ fB0PEA vtdEPeftse, SpOPY)P/ pE/jjW jQP15, Tf/CAOSpOfOf/ Sp., WLCkf.'P"AQPPEOfPE)~CeS CQPEQdef/SES, YQffON'/Q jtpOlp'/LCQ, gOQ SCBS mepef Qe RQd Z E&g0jtpOPf/VCeS jQC/0 SiWS.
Thc cxtTactlon methods described herein IQRy bc applied io csscQtlaHy Rny OlcaglnoUs rmcroorgamsm, The microorgamsm can operate, on„and/or live ullder any suitable coQdlt/ons& such Rs RTEacl'OblcaHy, RcToblcallly& photosynthctlcaHy& hetcrotrophlcaHy& R/lcll/Or thc llkc. According to cc/tain cmbodinlcnts„ thc yeast may bc cultured hctcii'otrophiicaHy ln thc QCC Of RIT, The tclTQ 'olcagIQous& Rs used hcrcln& 1cfc'ls to 0El bcal/ng, oil con'talQlng Rnd/Or produciQg oils, , fats, Rnd/or othe/' oil-like nces. OlcBglnous may Inchldc ol'ganlsxns that produce Rt least Rbout 20 pclccnt by vi;eight of Oils& Rt least about 30 pclccnt by weight of 0/ls& Rt Icast about 40 pcl'cent by vi'c/ght Oils& Rt least about 50 pclccxlX by %"eight oils, Bt least, about 60 percent by weight Oils, at least about 70 percent by v eight oils, at least about 80 percent by weight oils and/or th.e hke of the total weight of the orgamsrn Oleaginous mav refer to a microorgamsm during culturillg, lipid accuEQulation, at harvest conditions, and/or the like, Lipids suitable For use in production of biofuels may be extracted from a whole fcrmcEItatloEI broth cOQtalnlng Oll-rich nllcroblal ccHs of nous TMcroorganlsms, l/Eg to ccrtaln cmbodiiTkcnts, thc whole fermentation bl'0th. Quay include 8 sUgBI' fccdstock, FOT example the whole ferlnentatlon broth mav mclude about &0 to about 250 grams of hplds per hter of fermenter broth, about 0 to about 50 grams of' sugar per liter of felmenter brot, about 0 to about 40 gl'BTAs of salt pcf lltcl' of ntcf broth, BEEd about 10 to Bbout 100 gi'Bfrls of llpld- &ee dry biomass per ]iiter of fermenter broth. The oleaginicus microorganisms may include at least 40;~o bp weight fat, of bchvccn BboUt 40oo and about 80~o by weight fat, of bctwccn Rbout 50 Yo Rnd about /5 /o bv weight fat, 1IA. ccTtaln clnbodEIQcnts. 1 IEof to ihc ther lnal eatment. thc whole fermentation bfoih Inav bc pRstcuflzcd to EDRctlvRtc ccHuiaf cQzvlDcs RMII to climiARtc thc vlablllty of thc productioEI 01'gaDlsln to plcvcQi iicphcaillon Upon stoI'Rgc. r lzatlon also pfovldcs adcquatc control Encasufcs to EDEQEQEEzc damage io pf'oducEs of EA(Crest. En this case by also Enactlvatlng ihc llpascs. ihc pastcurlzattoA may be carried out by heating the whole fermentation broth to less than about 90"C, such as between about 40'C aAd about 80'C, fof less tban 3 hours„such as between about 1 minute and As mentioned„ tile RIDount of oil extracted map II3c IQCIcascd by catilng thc whole fermentation broth with heat, Pre-treatlnent of the whole feflAentation broth includes thermal treatment wilth coTECUITcQt chaDgcs iln process pH,. 'vvhlch ls Intended io cffcc't 8 thermo-chcIAEccll change in the cel! waH composiition. More particularly, by directly heating the broth to a teluperatufe greater than 90'C such as n about 90"C and about 150'C of between about 91'C Bnd about 150'C, or between about 100"C and. about 150"C, or behvcen about 110'Cand about 150'C, of between about 120'C and about 150'C, or between about 130'C and about 150'C, For greater than 3 hours, the cell waH structure undergoes ther lAal degradation, which es pefmeablhty ol the cell wall,. thus enabhng 011 to dlfkise more y during subsequent extraction ot a product from the oleaginous microorgMlisms. The exact Aature of change depends on ihe cell wall chemistry of the production strain. For Oleaginous yeasts„ Tclcasc of carbohydrates (nlonomcrs) colrlpf Ising ihe cell walls has bccn ed Rs 8 fcsul'i of tlEc plc-Ercatmcn(. Fof cxMQplc., holdiEIg ihc whole fermentation bE'0th Bt 121.'C for )ust ovcf 3 hours to BII30ut 8 hours with gcntlc silITEQg UslDg Ml lnlpcilcl EAay provide 80-85"./o cxtYBctablllty by slllg thc tv of thc cells. V. hllc Qot csscnEEal. thc systcIA IYIRy bc vcntcd to the Rtfnosphcl'c io facllltBtc coDccQtl'Rtlon 0f fcITDcQtatEon bl'0th fTQIA 80 /o dowQ to /0 lo water t via evaporation. '1'o nlinilnize tic activity, ii may be desirable to minimize the time spent by the whole fermentatiion broth coutainiug tbe oleaginous microorganisms between 45'C Bnd 80 C, This mlnlmlzatlon mav bc achieved by hcatmg thc whole fcflncQiaillon bfo'th contMQlflg thc olcaglD0Us DEECI'oofgRQisEns &om 45 C io 80 C En less thBQ 60 TAlnuics, 1D ccrtMQ embodiments, thc whole fermentation broth Lnay bc hcaicd Rt Ml avcIagc I.atc bchvccB aboUt 0.1 Rnd about 80 degrees Us pcl' minute.
During pre-treatment. the pll of the whole fermentation broth may also be ed, either by Rc!ding RD acid or 8 base. FOT' cxMDplc by fllst 8(lding Ml RCKI. Rnd then 8 base, this treatment may result in the early release of oil. Tlllrough the use of reagents and other aids, the pH Bmy bc adjusted to Rny lcvci within 8 range ol about 0.5 to 14, Using RCKis, bases, salts, or any conlbination of acids, bases, or salts. For examp/e, an acid may be added to adjust the pH to betv'een about 3.0 aDd about 6.0, As arlother exarnplle, a base may be addled to adjust the p1I:-II to between about 8.0 Rnd 8bout 10,5, hE ccrtaiLE embodiments, during ihc prc-treatment sRits BERy bc prcscDt EB 8 systcln containing thc whole fcl'mcntRtion bI'0th, resulting LB RB ionic strength estimated n about 0.01 M arid about 2.0 M in the system. The pre-treatment step is the Baost aggressive prolonged thcrnlal tlcatlncQt step EQ thc process RBd 8 majority of thc chcIDical reactions occur during this period, The subsequent mechanical lysis step releases the oil into a relatively mert, unreact~ve enviromnent. Fermentation broth that has been through the pre- treatment EDay be coalesced within less than 8 hours, suitably with 4 hours Of additional heating at over 90'C and mixing alone. JB rison, brotil that has been through pasteurization Rlorle EEEay e ive Rddlrional mlxlrlg, such Rs Biol'c than 8 ilouls of additional mixing„ar under 90'C to allow separation of an oil phase. ing to some nrlcBts„ thc Evholc fcrDEcIEtatlon bl'0th may Include 8 crude sugar source associated with sallts and ions at 8 concentration greater than about 0.05 g~L. As Used hcr'cm, thc tcITB crude sugar icfcrs to sugar extracts coBtalnlBg onc or morc dlsaccharKlcs or' BEonosaccharidcs dcl'lvcd fioIB coQEplcx rcncEvablc fccds'tock (including cMlc sweet sorghum. RBd sugar beets) ol corlccniratcd forirls of sugai extracts including sugar' juice„raw juice. thick juice„alld lrlolaSSCS. Crude Sugar can coDtalQ any combEBatEOQ Of diSaccharidCS and sacchal'Kics at gl'catci' than 15 wrro Up to 95 wt/o, with watcl', salts, LDEQcrais, fccdstock I'csidUc, Mid colrlplcx biomass forming thc dcr. Crude sugar nlay RitcH18tively bc desci'Lbcd Rs containing fr'oni 60 pro to 99 ro Rs 8 L'Rtlo of sugal' InoQomcrs io other solids ln thc dry matter. Tlllc other Q0D-sUgar' components of the (lry matter rnav Include sal'ts, nllQcI'Rls, ock residue„and complex biomass.
Thcsc salts Rnd ions associated with. thc crude sugM source Dlay include Na, K, CR, Mg, Zrl, Cu, Mn, Fe, Co, Rnd des, sulfates, phosphates, mtrates, RLEd combinations thereof.
These salts Rnd lons RI'c thus illtfoduccd Rt if RiloQS beyond those fcquifcd fol fcIYncntallvc growth of Xhc mlcx'ool'gRBlsnls, T4c salts and loQs Inay accumulate to 8 concentration ot 0.5 to 40 g/L, for exalnpie. Of particular uniqueness are potassium and calcium, w4ich may accumulate to 4Eghcl" conccntratlons t48Q Dlost 0't4cf elements Rnd Rrc different honl Usual ferxnentation broth media. These unique properties may facilitate the separation of hpid and water phases. MoTc pafXIcuiafly, 'ihc conccntrailon of potassnlnl E.s SUItably hlghcf Ehan thc concentration of sodium, 1ln ccftaln cIDbodlnlcnts, tlM coDccQXTRilon of caicnlm BIRy bc gl'cRtcl than l g/L. IIE ccTtaln cB'lbodllnents, the concentration of potasslunl mav bc gfcatcl' than 2.5 g/L, At the built-up tration. the introduced salts Rnd ions help with recovery of the oil phase by l0 a coalescence when the t oil is released from the microbial cells. Additionally at the buiilt-up concentration, the introduced salts and ions CHIBinate the need for addition of salts and ioQs oi(cn required to 4clp with lccovcfy of t4c Oll p48sc by coRlcsccncc, E,c,, mduccfs of demulsiFICTS. Thus„ the Eermentation brot4 may maintain thc same ion ratios or trations durlllg t4c coaIcsccncc stRgc, Rs wcH Rs during othcI' dovvnstEcaTB steps, Rs during 'iI'lc pl'c- tfeRXmcnt F0I' cxalnpic thc fclIBcntailoQ broth IBav 48vc R concentration of salts Rnd Eons of 0.5 to 40 g~L during coalescence.
Salts of Rddltlvcs to Improve cxtiactlon may bc added to 14c icfEBcntaXEQB of to thc was4 vi Riel Eox illlc sUgal souxcc, 01 to 40th ihc fcIIQcniatlon bfoth Rnd Xhc vi ash water for thc sugaf soul'cc, Along wlt4 i4c sRits and lons, Butx'lent fccds, crude of puflilcd Dutl'Ecni sources, mtrogen ox carbon, crude or paftiaHy refined sugar sources, and/or different v;Rter sources may 8lso bc added to tlM fcITQcntatlon media. %4CB carrying out t4e extraction method on 8, tation broth that inchldes a crude sugar a crude oil mav be recovered that ls lower En metals Rnd aruc ellements such as Na K, P, CR„Mg, Zn, and tiM ilkc, afcd to cxtTRctEon icchxllqucs that utlllzc whole dried bloxnass RIEd/of ts to fccovcx' exude oil.
Also during the pre-treatment, a pressure n about l0 psi and about i50 psi, , or betweef1 about 20 psi and about l50 psii„ofbet&veen about 30 psi Rnd about i 50 psi, or between about 50 psl and about i 50 psl nlay bc B.Ealntalncd ln 8 Q contalnlng tlM w4oic fermentation broth. This effective temperature and pressure Inav be k&wer if the system is held under a WO 95462 PC T/US2014/071055 FoIlowing the heating, the v hole ferlnentation broth may be cooled or dried, or both cooled Bnd drlcd, to RHow fur(hcr Isothermal (constant temperature) proccssmg. Morc particular'ly, lsothcrrQ81 pl'occsslng rcfcls hcrcln to ploccsslQg WLthout thc ILccd for addltlonBI hcatlng 01" cooling. %1th Tcspcct to thc cooling Rnd/or , for cxaIYlpIc., tile lvholc fcrErlcQtRELOQ broth MRy bc cooled to greater Ilhnn BboUt, 60 C, or grcBtcl" (hnn about !0 C, 0T greater than about 80'C, or greater than about 85'C, or greater than about 90'C. The fcrlYlcntatlon br'0th May also bc dcprcssurizcd, ln colnbinntion with the cooling„ to concentrate solids in the broth prior to further processing. r processing may include the apphcation of QlcchnQlcai dlsruptlon, UsLDg such dcvlccs Bs 8 bend QILH, 8 hornogcnlzcI', Rn orlhcc plate, 8 hlgh- 1.0 shear Tnlxcr. 8 press, Rn extruder, pTcssUrc dlsruptlon, wct g„dry g„oE' othei shcRT OI' LncchaLLLcnl dlsruptlon device for onc pass„ two passes, 01' Inol'c. Fol' example, Yw0 passes through a bead miH may provide greater than 90'N~ cxtractability. The further addition of acid LTEBy facliltaic coRlcsccncc. Tll'lc whole tation bI'0th Enny bc cooled Rt Rn Rvcl'Bgc I'Btc n about 0.2 Rnd nboul 80, or' bc(wccIL about 0.2 RQd about 1 dcgrcc s pcr MLELutc„ lor cxanzpic. AcklltloDRHy 0Y' BitcYTlatlvcly, 8 (lash evaporator mny bc Used 'to LTatc thc solids To provide Bddltlonal Bgltntlon, thc whole fcllncntntlon broth nlay bc stirred in 8 vcsscl Rt 8 tclnpcratuTc n BboUt !O'C Bnd about 100'C optloELBH$ including reflux tor be(ween BboUt 1 Rml about, 60 hours„ thus provldlng 60 to 85 lo 011 rccovcly, fol example. Jf desired, the whole ferlnentntion broth mny be held agitated at an impeHer tip speed between about 10 and about 300 CID pcT second, OT bcllwccn about. 120 BILd BboUt 240 cIQ pcI' second. This BgltatloQ may be can led out using any advantageous cornbmatlon of radial and axial flow lmpeHers, such ns Rushton or marine irnpeHers„ for example. QptionaHy„ fuller temperature adjustme~ts, pH Rdjuslllncnts„snit RdditioIL., or BQy colnblnntion of thcsc RctloQs nlny bc TELRdc duriELg thc ngitntioLL.
For exalnpie, up to about 2'N~ by weight of salt, such as NaCi, KC1, K,SQ4, or Na2SQ4, may be added to thc whole fcllncntRtloQ broth ln Ehe vcssci or alternatively Inav bc ed M SLfLI, for cxnlnpic„bv adding NRQH or KQH, plus H2SQg. As Bnothcl cxanlplc, Bn Beld ol' 8 base mny bc added to adjust a pH of the whole fermentation broth in the vessel to between about 3 and about 11. Thc heat generated fronl thc ation of acids Rnd bRscs llstcd Bbovc could also contnbutc to reducing the cQcrgy E'cqulrcd fol hcntLQg thc broth.
As Rn Bddltlenal pI'c-trcatIQcQt step, 't4c oleaginous microorganisms Inay bc sub(ected te lysis TcsultlDg ln Bn oil body Rn«i ccHular «icbrls partlclc slzc «ilstrlbution mi w4lch Bt least 80~/0„ or at least 95'lo of 8 volume of'released product cll bodies Rnd ceHular debris have a size r than 0.1 UIQ 111 er, wlt4 thc «llarnctcr' bclng the grcatcst ce across t4c ict, pRrtlcic, or body. The diameter may be measured using a Parricie Size Analyzer, available From Malvern (Instr«mlcnts Ltd of Worcestershire, UK. Merc particularly, r4c t4crrrlai pTc-tr'catIDcQt assists ln the lysis, vhich frees the oil once the biomass is digested. away. Due to this particle size lbution„ thc oil BIKi ccH dcbrls droplets Enay bc casllv Tccevcrcd 88 a. contlnuoUs p48sc t4r'ough sllllplc lnlxlDg cealcsccncc steps Bt BQ llrlpcHcr tip speed of gi'catcl' tiMD 120 cln pcr' second, on a 3-inch (7.62 CEQ) Rushton type impeHer, for instance. The coalesced lipid may I'csult ln 8 coRlcsccd. irprd paltlclc slzc drstrrbutren EQ w4Ich Rt least 80,~0, 01" Rt lcBst. 95/o, of 8 volume of cealcsccd lrprds have 8 slzc glcatcT 148Q about 40 unl ln dlalnctcr for lc A t may bc 8«ldc«i to clt4cT t4c dTy ccHs GI t4c iyscd fcrmcntatron broth, foHewlng thc 4catlng, to form 8 InlxtUIr'c. Exanrplcs of surtabic solvents Include hcxanc„(io«iccanc, dccRnc„ «ilcscl, Rlc040is, polar solvcrlts, Qen-polaT solvents, Rnd cornbnMtlons t4cr'cof, Thc Inrxturc Inay then bc c(l to RHow tl'lc solvent to contact Rnd cxtTRct thc 011 from thc wilolc ccHs of t4c oicagrnous elganrsms. Aftcl 8. surtabic pcrrod. Gf trme, thc stream can bc scpaI'Btc(i, such 88 by using 8 ccntrrfugc, scttllng tank, cvcioQc, 01' Bny cornblQRUGD of thcsc tccilmqucs, ie scparRtc thc solvent BQd t4c ell from thc fcrnlcntarron h. T4c solvent BQd thc 011 stream EQBy 't4cn bc reacted to CGDVCEC the 011 mlo a fuel component prior to convertmg the solvent and a rernamder of thc oil into 8 fuel coiYlpr'ising 8 blofucl. This nlctho(i of cxtr'acting liplds suitRblc ln thc production of rrncrobial OH results in an OH that is artificiaHy liower in metals„as the aqueous extraction process concentrates the metals in fermentation broth Rs compared to the eil by 8 ratio Of BE 1CRSt 2.
Rcslduai blorncal GT spent bToth I'csultlng fI'GID thc t4crrnal pre-treatment dcscri!3c«l 4cl'cln QMy ic hv«ilollysc«i ccH wBH polysaccharl«lcs Rnd plotclns ln Rqucous solUtlon, Encludrng mcdra Rnd gcncr'Btcd salts Rs wcH 88 dc-solventrzcd cell waH «lcbrls clthcr lvsc«i or unlvscd The al dellpidated biomeal or spent broth may be used as fertiHzer for crops, animal Feed, yeast extract, or 8 source of carbon. 'nutrients, for example. Mere ularly, due to the high lcvcls of potasslurn irl thc fcrmcntati'Ion broth, t4c spent brot4 IQBy l3c Tccvclcd Rs 8 petassluIQ source ED thc fonrl ot 8 lzcT for sugar frcids ol other clops. Using Bn 8«lucous pleccss, 14c WO 95462 PC T/US2014/071055 residual biamcaI may be in better form for these other potential uses coinpared to residual bioIDcal resulting fi'onl non-aqueous pl'occsscs.
F16. 1 ilhistratcs onc example of Bn Bcjlucous cxtracXLOYL process Uslrig tcEYlpcratuJ'c pl'c- treatment Rnd including the production of 8 yeast extiact. The process begins with a t'errnentaiion broth 10, ta which a base 12 may be added (optionally), While the fermentatiori broth 10 is heated LQ 8 vcsscl 14 ta 121'C Rnd held Bt. tllns cratLU'c fai' Bpproxlmatcip 8 hours„ an acid 16 may be added (optionally). Fo]lowing the heat treatme~t. the pre-treated broth 18 iis then coaIed (such as by flash-coohng, for example) to 60"C m a coolmg device 20 and iri thc process, water vapor 22 is released, Concentrate&1 broth 24 is then transferred to a centrifuge 26, whlcl'1 Xcs the broth 24 1Qto Rn 011 EYl 28 Rnd Bn aqueous cxtiaction residual sircaED 30.
Other types of scparBtion tcchniqucs, sUch Rs 8 sctthng tBnk ol 8 cyclone) mav also bc Used clther 810Qc 01 1ID coniblQatloxi with onc Bnothci. TlM BqUcous cxtlactloD lcsldual siIcanl 30 ls directed to a pressurizer 32 (or evaporator), from which water 34 fram. the pressure is released and a yeast cake 36 ls ci Bnd ded to 8 hydrolyscr 38 into which. Rn Beld 40 is added. 11M t Es 8 hydrolysed yeast cake 42.
Micl'oorganisIYls on which thc pi'occsscs lMlcED EYLRy bc carried 0Ut Lnchidc, IIMt arc Dot limited to, algae„ fungi, Rnd bactci la. Foi' cxainplc, 8 sultablc fuDgi IDRy c Oicagiiious yeast, such as those bclonglQg to tlM genus ol'LEla, PseLEdozvma, 01'Sj20E"IdloboILE5.
According ta ccrtaiQ cmbodLEDcnts„ thc vcast II3clongs to thc genus ApoxidioboiIEs pQE ai oseEE$. in 8 ic cmbodLIQcnt, thc disclosed DILcraorganism ls tlM mlcraargallism coLYesponding to ATCC Deposit No. PTA-12508 (Strain MK29404 (Drvi-13$)). lxi another specific cmbodiIYlcnt, ilM Enicraorganisni ls thc microorganism corrcspondlQg to ATCC Deposit No. PTA-12509 (Strain MK29404 (Dryi-182J)). Jln another specific embodiment, the micioorganisEYi is tiM LnicroalganislT1 ponding to ATCC Deposit No. PTA-12510 (Strain MK29404 (DI'y1-173N)). in Rnothcl' specific cnlbadilnent, thc microoi'ganisln is thc microorganism corresponding to AYCC Deposit No. 511 (Strahl MK29404 (Dry55)). in x specific cLYlbodimcnt, thc microorganism ls 'thc rganism coxrcspoQding to ATCC Deposit No. PTA-12512 n MK29404 )). in another specific embodiment, the microorganism is the microorgamsm corresponding to A II. CC Deposit No. PTA-12513 (Strain MK29404 (Dryi)). irl another specific embodiment, the microorganism is the Lnicraorganism correspoiiding to ATCC Deposit No. PYA-12515 (Strain MK29404 (Dv;1-]47D)). in another specific cmbodlnlcnt, , thc lnlcrooI'gRmsnl ls thc llllcroorganisxn corresponding to ATCC Dcposlt No. PTA-12516 (Strain MK29404 (Dryl-72D)).
Yeasts have polysaccharide ceH walls to protect them froxn environlnental stresses, such as shear forces, osxnotic imbalances, predators, and the Hke. The protective ceH wail can make it difficult to harvest intraceHU)ar BletabO)ite, such as Hpids in nous yeast that can be convcrtcd EDto blofUcl.
GlycosEdic enzymes Rrc useful ln ing down polysacc)Ml'ldcs, Rnd thus for dcgrad)Qg yeast ccH waHs. idic enzymes RI'c often Rctlvc OQ tl'lc specific sugar IQ0BOIBcI's within 8 po)ysaccharidc, and thc spcclflc llnkagcs bctwccxl IDOBonlcx' sugars. For 1BstRBcc, glvcosldic cs can differentiare bctwccn EI-)-4 )EBkcd glucose se) and. p-)-4 llnkcd ghlcosc osc). However, yeast RI'c not RH colnposcd of idcntlcal pollysaccharidcs but rather differ Widely % 1th TcspcE. t 10 thc type& End s of sac,chhrEdc monomer% And thc types Of )Inkagcs bctwccB Dlonomcis.
Conscljuent)y, thc mixture of glycosK)Ec enzymes th8( Rrc ) for ceil waH dcgradatlon ls dependent on thc organlsQ! OBc partE ular oicagiBous yeast used ln ting sugar-to-dicsc). SpOFIdlo/Io/45 paPTIFosells MK29404Dry), has 8 particularly Dove) ccH WRH structure. A common structural linkage in Inany yeast is p-) -3 giucan. )Ifowever, MK29404Dryl showed on)y 8, )Ett)c 1-3 linked glucose. Rnd 1Qstcad EI4 glucose was thc ma]or sugRT llnkagc. r cormnon component of yeast ceH waHs ls , which Es often colnposed of 1-6 )inked e monomers. Iln contrast„MK29404dryl contains very )ittle )mannose, but rather contaiins both 1-3 and ) -4 linked mannose.
Bccausc of thc partlcu)8T composltlon of thc MK29404DTyi yc8st ccH waH. 8 speci)lc combmatlon of enzymes ls needed to effectively degrade the Inlcrobmi ceHI wail, )t has been discovered Xhat 8 con'lblBatlon 0) cBzyQlcs Enc)udlng Rxnylasc 1-4 maIEIEosldasc„and 1-3 mannosldase ls particularly effective m breaking down oleagmous ceH wallls of oleaginous microorgamsrns that include MK29404oryl. )n palticu)ar„amy)ases specific for o,-)-4 linked g)ucosc 81'c alllv cffcctlvc. Fol' cxalnp)c, thc colnblnatlon of s lnay Enc)udc bctwccn about 5 /o RBd about. 30/i), ox' between aboUt 6/o and about 25 N&, 01 bctwccn Rbou't 7 ro and RboUt 20oo II3y wclg)Et RIDylasc; bctwccn about 5'io and about 45'Zo„orbctwccB Rbout )O'N~ Rnd about 35,o, 01' bCtWCCQ about ) 5, o Rnd about 30 o by WClghX 1-4 mannosldase; alld bCtWCCB about 5,4) and about 45 Zo, or betv een about 10'/e and about 35 80, of between about 15'/o and abcut 30'Io by weight 1-3 Mannosidasc.
Ykc comblnatioQ of cs IQay also include onc of Morc afy lcs„such Rs pTotcascs, SBHRtascs„chitinascs, of arly coMblnRtion ot thcsc cnzvincs to Improve cEEzyYIEc performance and lipid recovery.
EEtkci" priol' to of subsequent to Using thc combination of cnzyYQcs to bleak down thc oleaghlous ceH waHs within 8 whole tation broth containing oleaginous microorgaEEisms, tke vihoic fcYYQcntatlon broth Enay bc ihcfmBHy pfc-treated Bs bcil above. Morc particularly tke broth Inay be heatedl to a temperature between about 90'('and about 150'C for Inorc than 3 hours.
After using the combination of enzymes to break down the oleaginous ceH waHs v ithin a whole fcITEEcn'ta'tlon broth Qing olcaglnoU!s Microorganisms, intfaccHulaf TnctBbolltcs may be harvested from the oleaginous ceH. waHS, The intraceHular metaboHtes suitably contahi hpids.
Tkc cxtfactcd lllpEds IQRv bc Used EQ 'thc production of biofucls, such Bs bio-dci'lvcd dlcscl.
Moi'c pafticB18flv Rs dcscfEbcd ln gTcatcf detail above 8 solvent s'Bch as hcxanc dodccailc„dccanc, cllicscl, Rlcohols, 01' Rny coinblnRtion of tkcsc solvents, may bc added to thc dFy ccHs or lyscd tation bI'0th to form R Tnixtur'c. Ykc mixture of ihe broth and ihc soivcllt may bc agitated to coDtact Rncl cxtfRct oil from tkc olcaginolls vcast ccHs. Yhc solvcQt Bnd thc 011 May uently bc scpRlatcd from thc bloth, suck as by Using 8 ccQtfifugc. ihe solvent Rml thc 011 Dlay bc reacted 'to coDvcfr Bt least 8 portion of thc oiil into 8 tuel ncEEt. Yhc solvent Rtld 8 fcinaindcE of the oil May bc convcItcd 1Dto 8 fuel, DRFAcly 8 biofuei. Yhe spent bFoth Tnay bc used Rs fcitlllzcI foI crops, RQEMal feed. yeast extract, yeast kydIolysBic, of 8 source of carbon/Qutrients.
As described in gl'catcT dctalll below, Bny RElueous cxtI'Rcnon cÃucnt fcIQBEDEDg Bftcf hav~esting thc lntIRccllulaf Mctabohtcs Inay bc lccyclcd. I'or cxanlplc, tile Fccycicd cxtlaction v ater may be used as imbibition water for washing a process feedstock to extract sugar, &16. 2 Es an. Integrated to-dicscl flowshcct showlQg kow us extraction effluent fcinainiEEg after lipid vall ls cd to thc front-cnd sugar IccovcIv opcratlons.
More paiticulariy, recycled extraction water is used as tion water tor wasllling the process feedstock to extract sugar, Suck integrations are beneficial because a greater yield on feed materials is reahzcd, as well as a reduction in waste management capital and processing costs.
While lt ls always of lntcrcst to rccvcic waste strealns, the kcy Es ldcntlfyiing thc proper rccyclc poilnt within tll'lc Aolvshcct that maxiTQizcs I'ccovcry value, while also Bccoullting foI' how rccychng Rffccts thc dynamics sind optimum operation of thc lnlcgratcd ct.
As dcscllbcd Bbovc. sugar may bc tcd to blofucl„ lncludlng dlcscl for cxaEnplc, Using heterotrophic organlslns wi'th Bn BqucoUs cxtracXion section„whcl'cby thc prodUct Ilrplds BI'c removed and recovered diirect1ly from the aqueous ferlnentation broth. Product is recovered from EntcHlal coxnpRrtrnents of thc lnous olganlsrrl bv coil'Qblnatlons of al, Incchanlcai, osmotic, Bnd enzymatic forces, resultillg in a multi-phase t stream that contains less dense 1lipids, residual broth water, and dated biomass, As iIlustrated in FlG. 2„the residual broth l0 waXCT can be recyc1led and Used as imbiibition water for v ashing a process feedstock to extract Thc AIowshcct in FlG. 2 shows sugM'canc l00 Rnd lnlblbinon water f 02 fcd to 8 mIH f04.
From thc mill l04„8sUgRI' solution l06 ils fcd to 8 trcatEncnt dcvlcc 108, whllc bagasse l l0 ls tcd GUt. FT'OTQ thc trcatnlcnt. dcvlcc l08, 811 MFV (multi-effect evaporator) fccd l l2 ls sent l5 to evaporators 1/4, while mud 1 l6 iis separated out. From the evaporators l14, a vapor/gas strcBTQ l 18 ls fcd to 8 sccd fcrIQcQtatlon dcvlcc l24„whllc 8 collccxltrRtcd sugar strcalxl l20 ls fcd to 8 TQMQ fclTHIcntatEOQ device l26„8Qdwatcl' l 22 Is scparatcd Olilt. Along wrth thc tl'Rtcd sUgar stream i20, Mr l28 iis also added to thc xnaln fcEDlcnXRtlon dcvlcc l26. FroxQ the main fermentation dcvlcc l26, broth l30 ls fcd to Ml Bqucous extraction dcvlcc l34 while watcil" vRpor 0 BTId CO2 l 32 Rrc rclcRscd. FXGIQ thc aqueous cxtractloEI dcvlcc 1 34, 8 product oil 136 ls scpalRtc(i out„evaporated watcl l38 ls rclcascd, RQ(ll waste water l40 ls rccyclcd Into thc imbibitiorl water strealn l02. TaMe l shows sanlple Aow magnitudes for major s and components ln thc sugar-to-dlcscl Aowshcct of FlG. 2. Based GQ ihc datR ln TRMc l, Rn EIQlblbatloQ iwatcr rcductloQ of 40 E(3 ls calculated vlth this rf Q Bttrlbutabie to rccycllng thc waste water. AdditlonaAy„a solid supplement to bagasse Of 5'/o is calcUiated, also attributabie to Eng 'thc waste v'Riel'.
TRblc l: FkNv udes tor Major Strcanls Rnd CompoQcBEs in the Sugar-to-Diesej Flowsheet Total SugRI'CRTEC linbib water 300 300 l0 j0 MEV feed 904 . VG steam CoDC sugar W'Rtcr Broth :;'6'aste water l.386 ing tl'lc v'Rsic water Rs 8 portion of thc EnlbibEtloQ water gcnciatcs Inultlplc unexpected benefits, including: Recovery of organic carbon that can be father converted to product (sec bcilcist 5) 2. At least partial I"ccovcI'y ok organic Rlld EBorgaEElc Dutrlcnts from non-hpld blolnass and redu. ccd hrst intent nutrient feed (e.g. Rminonia) 3. ScpRrailoB of Unlccovcrcd non-lipid bioEnass trom sohltlon by coiningling with bagasSC 4. Additional boilcT tccd Rnd cnci'gv gcncI'ation by cominghEEg bRgRssc rnid UIErccovcEcd Don-lipid bloTBRss . Rcduccd sir'Engcncy on lcrniciitation opciatloQ RHowlng for glcatcl' organic cRrbon slippage Rnd I'ccovcry by rccyclc (scc bcDcflt l) 6. Optimization of. sUgar str'caIBs l06 RDd l20 for usc Rs specific feed strcains to thc seed Rnd maEB fclYDclitcrs 7. Reduction M fI'csh watci Ed by UBEBg recycle watcT foi' Embibltion 8. Reduction EQ capital Rnd operating costs for waste cnt The e streains Inay be implemented in the previously-described methods to Emprovc thc rccovcTy Rnd convcislon of kcy tuents Rnd Improve overall cHEcicncy, Foi' cxamp1le, in a method of extracting hpids le in production. of biofuels from an aqueous fermentation broth, whcfclQ thc broth. contains olcagilQous fganlsrQs 0Y' sUgaY'canc„of both olcRglnous microorganisms Bnd sugar'canc, thc aqueous fermcnration broth DIRy bc pasteurized, such Rs by heating the aqueous fermentation broth to about 40'C to about 80'C for about l Dllnutc Up to almost hours Thc us fermentation broth may bc thermally pl'c-tTcatcd bv heating the broth at a ature between about 90'C Rnd about l&0'C, or be(ween about l00'"C and about l &O'C or behveen about l l O'C and about 1~0'C or between about 120'6 and about l50"C, or between about l30'C and about l50'C for about 30 Eriinutes to about l 8 hours, or more than 3 hours te about l8 hours, or mere than 3 hours te about 8 hours. The aqueous fcfiiientation broth may bc stlffc(i during thc heating val. An acid, 8 bBsc, of beth BQ acid l0 Rnd 8 base lnav bc added to thc s fermentation broth, Thc aqueous fcfrncQtatlon lieth may be passed through a bead mill of other ical device at least once, or Rt least twice, of moTc. Thc RqucoUs fcfmcnration bforh H18v bc siiirfcd m 8 vcsscl Rt about 70 ( to about l00'C or u~der reflux for about l to about 60 hours, A salt suchas up to about 290 by weight of the sR1lt, such Bs NRCl, Kcl, K2SOg, Oil N82S04„iriay bc Rddcri te thc aqueous fcfmicntatieQ broth ln l5 thc vcssc1I of alternatively Inav bc produced En.%EPEE, iof cxainpjc, by Rddlng NROH ol' KOH, phls H2S04, AD acid 0T 8 base may bc added to Bd)ust 8 pH of tIic s fcnrlcntatlon bleth ln thc vcsscl to bctwccQ RboUt 3 and BboUt l l, Thc llplds il'nav bc separated f loni thc aqueous fcrnlcntarien broth thl ough Bn Bppr'opfiatc sohd-Ilquld-liquid scpail'Brien schen1lc thRt. may Enclludc one or more steps such as gravity separatiori, hydrocyclones, filters, Rnd!Or centrifuges, leaving biomass se1llds and residual broth water, The residual broth water can be used as lmbibitioii water for washiing the process feedstock to extract sugar. Additionally, the biomass sc Hds can be recycled with the residual broth water.
Thc llplds Dray bc convcftcd lnt0 8 bio fuel through. thic Usc 0f hydi'otf cating el' sterification„ fer example.
According to ccftMD cmbodiinenis„ thc invention Inay bc directed to 8 EnanufactufiQg fRcllllty foi' producing blofucls. According to ccftMQ CEnbedEDICEIts, rhc cturing lacillty may include a lipid tiion unit. Additionally, the cturing facility may include a thcrnial pic-tfcatIDcQt UDEt. lD ccftMD cnlbedilnents, thc nranufactufing facility IDRv 1Qchidc cqUlpmcnt thRt enables Tccyclc of I'csiiclUal bi'0th wRtcI'.
WO 95462 PC 14/071055 AccoFdlng to certain clYlbodilncQts„'tiM invcEIYEGD Diay bc dlfected to 8 renewable IDBtclial or 8 blofuel, or both a renewable al and a bic fuel, made according to any of the methods described herein.
According to ccYtRin clnbodimcnts, tlM IYlcthods dcscIEbcd hcrcln may TcsUlt ln Rrl lncTcasc En thc oil cxtfacYion yield ot thc organism Fof cxRIDplc tiM nlcthod Diay 1csUit ln an Increase ln tlM 011 cxtf'acitlon yield of thc mlcroofgaliism of at least about 10 lvclght pclccnt.
According to certain embodiments, tlM lncrcasc ln 011 extraction yield lnay bc Rt lcRsE about 10 weighY pclccnt, at. Icas't Rbout, 15 weight pcfccnt GI' RY least about 20 weight pcrccnt.
Examples OQc metric Used Yo chalactcrizc QM performance of thc cllcscribcd lnicroorgallisEDS ls fatty acid cxtractability, OT PAE. TlM FAE of BEYS of thc DHcroorganisms according to thc dlsclosuFc can bc calculated accofdlDg to ihc following formula: ! e:, 00- c,.
,, „,.„,~(I. .. ) ' I (,.'.„,,„sX(&00-&, bjot!.ass wherein b is the total. biomass after ceil Ewpture, typically measured in grams; ~ j!.'omoss ls thC pCE'CCntagC Of F AME prEOE to cCii rupture& Wherein t ss ES caicuiRYCd Bs total gEanis FAME ovcl 'total grREDS s, " thc tcfID !FAME, as Used hcl'ciQ, refers to 8, fatty acid methyl ester„ Cgkjop&!soj ES the pCI'cCntagC Ot 1 AME RftCI' CCii rupture~ WhefCEYl Cpjo~oo/ 1S calculated as toE81 glanls FAME ovcY' total gFRDis blolYlcal; Rnd 3 Es thc total Inass of oil after ceil Y'c, but priof to thc oil Tccovcry step, llly measured 1Q grRnls. Obtaining tlMsc values from lhc n'HCToolganlsTQ GI' fermentation broth Es within the ability of olle of ry skiiil in the art.
According to some embodiments. the methods described herein may result in an increase ln ilM 011 of fatty acid extract'Eblilty index of thc microorganism FGT cxaIYEplc, thc Dicthod Qlay fcsult Hl Rn ase ln thc FAE index 0$ thc microorganism ot at. least BboUt 10 weight pcrccQt. ln certanl embodiments, foliowmg oil recovery With hexane„ the Ynass of the oil ls measured (L). Also measured ls the FAME after 011 recovery with hexane. ln some cmbodinicnts, vacuulYE evaporation, as ls known IQ thc BYt, is pcl'forIYEcd On tlM sanlpic prior to Thc extraction yield of Rnv of (hc microorganisms RccoTdlng io thc disclosure cBQ bc calculated according to the foHowing formula; whcfclQ 8 18 thc total bloTQass pHQT to ccH rupture, typlcRHy measured ln ' grams, ~ Liornccss ls ihC pCECCBtagC OtFAME prlol io CCH rupture. . WhCTCln Cg Omass Es CmlCUiatCd 88 total grams FAME over totRi grams biomass; C ls thc pcI'ccQtmgc of FAME Bftcf ccH Tupiul'c Rnd oil Tccovcl.'v whcl'cEB C 18 i0 calculated by total gfanls FAME Over to'iai cJTams oil ~ 8 1 ls thc to(mi IDRss 01 oil after ccH rupture BBd oil lccovcry 'typicaHy ed lB gfBMs Obtaining these measurements from the rnicroorgamsm or fermentation broth is within the abiLity of one of ordinary skiH in the mr(, Accoiding to some embodiments. thc Incthods described herein Mmy result EB RB lncfcmsc i5 IQ thc 0H extraction vicid of thc microofgmilism. Fol cxminpic, thc Incihod Inmy fcsuit ln Rn lnclcasc ln thc oil cxtI'action yield of ihc Microorganism of mt least, about i 0 weight pcfccnt.
Kxmrnpic I. FcnflcnltmiEon 01. Rn nous vcast stl'BID Using 8 conlplcx sUgRf source that included sugar juice yielded Unpasteurized whole broth for furtlier processing. The whole bl'0th wRS pasteurized bv g En 8 vcssci ff'onl 27 C to 80'( ln 30 Enlmltcs hcM mt 80 C fol' 3 hours The pasteurized broth «howed a fatty acid extractmblhtv (FAE) Ot i6 8'0 l«'0 oil phase was recoverable upon centrifugatlon oi the pasteurized un-iysed broth ln 8 bench fuge at 4500rprn (4000g) for 5 minutes.
AB aliquot of tile pmstcuf ized bro(h was prc-treated ior 8 houi'8 Bi 106 C ln 8 20L Jacketed tank fitted with 2 Rushton, rs The tenlperature increase from 26'C to i06 '( o"curredl ovcl" Rbo'Ut 45 cs„mt, 8 fate of RboUt i.g C/IQEQ. ihc prc-treated broth showccll Rn lncl'case ln fatty acid extractabiiiity '/0). No oil phase wms recoverable upon fugation of the pmstcurizcd Un-ipscd bloth 1EE 8 bench centrifuge Bt 4500Tpnl foi 5 cs. Results Bfe plcscBtcd in Table 2.
Paste)tria. eoodttiotts Pt e-treatmeot Coaiese. Ce))tri fit 8. dAtlloa s Co))ditto))s eoEldAtloB% Extra No. et ot 'E iate Beooh Yield rate, Pass iro s~cIoa tY.'i.'o) es 8 i.02 Y'! )011)))',I, sl! 80 91,51 pEI 6.31 Wltoi)e broth .40h 4000a 69.89 tat'!o =- 38.03, dr' No basis 8(i )06 Eree 80.76 o l.l - tt- sti 106 ox 53 -'16h 4000$& 5 odtt 4000a 5 otitt 84.1 ).3 The rized Bnd catcc1I bio th wcI'c each. iyscd Bt varying Bow 1'ates, 80mi/TxilButc oT 380IDl/IYliDutc fol' i or 2 PBsscs EEI 8 KBL PEioi bead-miHl (i.4L vessel filled to 85zo f11li vollunic with 0.5mni slllca-zlI'coQlR Enedia). ihc pretreated bloth exceeded thc fn'ttv acid cxtl'Bctnblhty (FAE) of thc pastcullzcd broth with mai I'csK!cncc tlinc ln thc IDEih Thc cxtlactablll'ty of plctrcRtcd. broth ivscd fol i pass Rt thc highest speed (380m1I/TMB) was comparable to the extrnctabiiity of the pasteurized broth when pmcessed at lowest speed i 0 (80mi/min) fol miLdtlpie passes.
Sample (200-300g) of pasteurized or pretreated iysed bmth with fatty acid extractabihty --95'h~ was adiustcd to pH 4 using 3N suifuY'ic Beld. Thc sRFnplc was coaicsccc1l ln batcll Inode under TcQux (500 mi &zicnmeycr flask with stir-bar). Coalcsccncc was IDonitorcd 113y fugation of i 5-50mi aliquot Bt 4500Ypm (4000g) Eor 5min in n bench centrifuge.
Coalesced broth upon centrifugation showed a distinct separate oil layer with a lower layer sing spent bmth. Coalescence of the pre-treated iysed broth was completed within i6 hours. The pasteurized lysed broth required over 40 hours to coalesce.
The oil layer was recovered froxn the top of the centrifuged U&es to estinlate extraction yield. The extraction yield for the eated broth was 84, 1',4 while that cf the pasteurized broth was 69.9 ro.
Thc 011 quality is coQ!monjy dctcrlnlncd bv lcvcj of frcc fatty acids (FFA) v18 loD, Tl'lc fl'cc fatty acid lcvcl 1Q crude oil I'ccovcrcd froln both 'thc pasteurized bI'0th and pic-treated bmth. were sixnilar. (1.2- 1.3'io) Example 2. FCETncntation of RB ojcaglBous ycRst straiB Usnig 8 conipjcx sugal source that included sugar juice yielded unpasteurized whole bmth for further pmcessing. A flow diagl Bm of tjxc process of th] s cxRIDplc ils Illustrated ln FiG 3 A s shown ln Fio Unpasteurized whole broth 210 was ted using 8 protocol that. Included pastcurlzatloB of thc bmih 210 in an agitated jacketed vessel 214 at 8(j'C for 3 hours, The pasteurized broth 216 was then M1ljustcd to pH 4 usilng lc acid BBd subjected to 8 pl'c-trcatnicQt phBsc 218 Bt 121'C, re of 30 psi (15 psig) fcr 8 hours. A temperature ramp of 1.8'Cimin was used for the heating of bl'0th; broth was cooled at 8 rate of 0.23 C/il'BEB. TIIM prc(Teated bToth 220 wRs thcil sub]ected to 8. lysilr!g phBsc 222 uslBg 8 nllll Rt 00 Q11/DEED foT 2 passes. Yhc lysccl. bil'0th 224 was Ellen sub]ected to R coallcsccncc phRsc 226 ln 8 wc[i-agitate(I) tank Rt 90 C, 70 Io xnolsturc. Thc coalesced broth 2 8 was then ted to 8 solid-jlcjuid tilon pllasc 230 iln wllllch thc coajcsccd bloth 228 WBs centrifuged through tavo-phase Bnd thrcc-pllasc ccntrlfugcs to il ccovcl' thc crude 011 232, Yl'lc process also ylcldcli 8 spcQt bl'0th phase, wlluch was sepal'Btcd out Gl'to 8 spent hcavy phBsc 3 il, alld Dlultlplc assorted solid lns 236.
Tile conccnllTBQQQ of Dictals EQ 8 sanipjc of thc Unpasteurized whole brotj1I, in thc recovered 011 Bnd ln cRcjl of tlat exit strcanxs EEIcjuding thc spent hcavy phBsc REId tllM solids was analyzed bv K P analysis. TlM I'Btlo of concentration of Glctals EQ unpRstclirlzcd whole broth Bnd ln tlic recovered crude oij shows that ihc startEng whole . had Rt least twice ihc coQccntx'RtEQD of Bictals (cxcjudlng Sl REId Cu) than thc 011 I'ccovcrcd fronl thc pl'occss. Yhc crude 011 rccovci'cd froin tlat process was icantly depjctccll of Na, Mg, P, K, CB, MEI, Fc, RBd Zxl Rs compared to tlM whole fcrnicQ'tatioQ bE'0th.
Thc crude 011 rccovcEcd froxn thc cxtl'RctioB pl'occss was also signifMantly dcplctcd of Na, Mg, P, K, CR, MQ, Fc, Rnd ZD coEnparcd to thc other stlcanls that exited the process such Bs thc spcQt hcRvy phase Bnd tjM sojEds following extraction.
TRbic 3: CoIDparison ol Concentrations of s ln %"hollc BI'0th to Crude Oil Recovered floID Aqueous tion Process ', P I K l Ca MB' ,:Fe I Cu SREElplc Nalnc' , (33%4'L ] I [3318/1. j iDElL;, !E&vgl't, lit, ET. EI Intp„l. , ; (B34ri. 3. Ln!g/L fm~, '(. I', Lmg/L l I I UtlpastcuE'1zcd wsdlolc ill'o h ,'EO18,:486 22 I 0617m)3) I Spent. )leavy phase "603 :;606 83; 352, 680, l Y()03 .', (MN0827)3-2 N2 So)tds 3'oEE3 ( entltluge (/) .') ' 9 )0 36 I 33 / I 4238 l 2661 l (MN082713-3 N2 I 0617EB)3) Soltds fron3 rttge (i)3) 1474 60 426 6481 l 2292 ;'(MN0827) 3-4 N2 0617m 'I 3 ) I Crude oil 26 0:, 283 l 63 0 l (SR) 00) 13-13 N2, 0617EBl 3') ntratlon H.atlo, 11, 19 UEEpastcuri:. cd xvl'Eolc )3rot)E to Crude oil Concentration Rat!0, Spcttt 23 18 1', l 7 l 10 hcav3' pllasc ()'Eius«1) to Crude oil Collccntratlon , sol!ds 21 I 36 10 I 18 l 639 :;lionl centrifuge (//4) to Crude oil COAecntratloB Ratio, so.'ids ;'li'OEB cct!trifuge ((65) to I Crude oil KxalrEpie 3, %hole lcrIYlcntation broth ot Rn oleaginous yeast strain vt/Rs hcatcd to l2 l C in BD agitated vcssci for 4 hours. Thc broth v'Rs thereafter cooic(1I to 60 C and iyscd through R bcadI mH. li (KDL Pilot„Glen Miils, NJ') mn at 3 different flov" rates respectively (380 , 200 mi/min. Rnd 80 mi/min) to rcicasc intraccHular oil product. Pal chicle sEzc dlstnbutioQ of I'clcasc(i oil Rnd ceil debris foHOEviing lysis in the mill is shovYI in FlG 4. AH measureabie volume of Ilvscd cciis Rndl oil dlroplcts cxcccds 0.i Enicrons, EQdlcatlng to tltc potcntEai to usc proccsscs such i.0 Rs centrifugation to separate Rn oil and solids phase upon further processing.
Thc oEl product ln thc oil flactloD can bc recovered by O'Dxlng Rt 80 C or higher temperatures. 5L each of broth iysed Rt 380ml/m v/as placed in a vesse1I mixed vviith tEvo 3-inch (7.62cm) n impeliers. The lysed broth v/as mixed ai agitator speedl ok i 50 rpm (tip speedl -- 60cm/s, tovl/er NBSi6') or Rt Bn agitator speed of 500rpm (tip speed =- 200 cm/s, TO&ver l5 NBS i 7). Thc disitributloQ of oil RDd ccH debris at thc cnd of 6 houls of mixing ls shovY! En FK».
. PI'oduct frolD thc vcssci Rt 500EpED, tip spccd of 200 cITh s, showed R distinct scpar ate oil phase upon centrifugation at 4500 rpm (4000 g) for 5 minutes in abench top centrifuge. Product &oln the vessel at i50 rpm, tip speedI 60 cm/s, show Qo &ee oil and demonstrated an emulsion phase upon centrlfligatlon. it wiH be apparent to those skiHed in the art that various modifications and variatioIES can be made in the diisclosed structures and Inethods without departing from the scope or spirit of the lnvcntlon. Particularly, «lcscI'lptlons of RQy onc cmbodlnlcnt can bc &cclv combined with descriptions oI other cmll3odlmcQts to result ln conlblnations Rnd/or lons ol two or morc ts or Hmitations. Other embodiments of the inventiion wiH be apparent to those skHllcd in the art from consideration of the specification and practice of the ion osed herein, it is i0 1Qtcn«lc«i that thc specification an(l cxalnplcs bc considered exemplary only„with R true scope and spirit of the invention being indicated by the foHowing claims.
Claims (1)
1. A method of ting lipids le in production of ls from a whole fermentation broth, comprising: 5 (i) pre-treating the whole fermentation broth by heating the broth to a temperature between about 90°C and about 150°C, or n about 100°C and about 150°C, or between about 110°C and about 150°C, or between about 120°C and about 150°C, or between about 130°C and about 150°C, wherein the broth contains oleaginous microorganisms which are oleaginous yeast cells; 10 (ii) subjecting the oleaginous microorganisms to lysis, resulting in a droplet and debris particle size distribution in which at least 80%, preferably 95%, of a volume of released product oil droplets and debris have a size greater than 0.1 um in diameter; and (iii) subsequently extracting a product from the oleaginous microorganisms; n step (ii) further comprises recovering oil and cell debris droplets as a 15 continuous phase by mixing at an impeller tip speed of greater than
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| MX350779B (en) | 2000-01-19 | 2017-09-18 | Dsm Ip Assets B V * | Solventless extraction process. |
| EP2576801B1 (en) | 2010-06-01 | 2019-10-02 | DSM IP Assets B.V. | Extraction of lipid from cells and products therefrom |
| US11124736B2 (en) | 2013-12-20 | 2021-09-21 | Dsm Ip Assets B.V. | Processes for obtaining microbial oil from microbial cells |
| AR098890A1 (en) | 2013-12-20 | 2016-06-22 | Dsm Ip Assets Bv | PROCESS FOR OBTAINING MICROBIAL OIL FROM MICROBIAL CELLS |
| KR102435269B1 (en) | 2013-12-20 | 2022-08-22 | 디에스엠 아이피 어셋츠 비.브이. | Processes for obtaining microbial oil from microbial cells |
| NZ721409A (en) | 2013-12-20 | 2022-10-28 | Dsm Ip Assets Bv | Processes for obtaining microbial oil from microbial cells |
| AR098896A1 (en) | 2013-12-20 | 2016-06-22 | Dsm Ip Assets Bv | PROCESS FOR OBTAINING MICROBIAL OIL FROM MICROBIAL CELLS |
| US11259527B2 (en) * | 2015-08-17 | 2022-03-01 | Heliae Development, Llc | Haematococcus based compositions for plants and methods of application |
| EP3180990A1 (en) * | 2015-12-16 | 2017-06-21 | Neste Oyj | Method for preparing a composition having antimicrobial activity |
| US20220154098A1 (en) * | 2019-03-14 | 2022-05-19 | Dsm Ip Assets B.V. | Methods of obtaining lipids from a microbial cell composition by enzyme and ph shock |
| EP3983509A4 (en) * | 2019-06-14 | 2023-05-31 | The University of Melbourne | Shear-induced phase inversion of complex emulsions for recovery of organic components from biomass |
| CN110734867B (en) * | 2019-11-29 | 2021-04-20 | 自然资源部第三海洋研究所 | Deep-sea-derived yeast Q7-7 and application thereof |
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| GB1466853A (en) * | 1973-05-22 | 1977-03-09 | Simon Rosedowns Ltd | Extraction |
| WO2010120939A2 (en) * | 2009-04-14 | 2010-10-21 | Solazyme, Inc. | Methods of microbial oil extraction and separation |
| EP1178118A1 (en) * | 2000-08-02 | 2002-02-06 | Dsm N.V. | Isolation of microbial oils |
| DK3111767T3 (en) * | 2002-06-19 | 2019-08-26 | Dsm Ip Assets Bv | MICROBIAL OIL AND PROCEDURES FOR PROCESSING THEREOF |
| US8470172B2 (en) * | 2007-01-09 | 2013-06-25 | Siemens Industry, Inc. | System for enhancing a wastewater treatment process |
| US8080684B2 (en) * | 2007-06-27 | 2011-12-20 | H R D Corporation | Method of producing ethyl acetate |
| UA109884C2 (en) * | 2009-10-16 | 2015-10-26 | A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD | |
| UA111708C2 (en) * | 2009-10-16 | 2016-06-10 | Бандж Ойлз, Інк. | METHOD OF OIL REFINING |
| US20110252696A1 (en) * | 2010-04-14 | 2011-10-20 | Solazyme, Inc. | Fuel And Chemical Production From Oleaginous Yeast |
| EP2576801B1 (en) * | 2010-06-01 | 2019-10-02 | DSM IP Assets B.V. | Extraction of lipid from cells and products therefrom |
| SG10201509035WA (en) * | 2010-11-03 | 2015-12-30 | Solazyme Inc | Microbial Oils With Lowered Pour Points, Dielectric Fluids Produced Therefrom, And Related Methods |
| EP2867355A1 (en) * | 2012-06-29 | 2015-05-06 | BP Biofuels UK Ltd. | Process for separation of renewable materials from microorganisms |
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